Epidermal Growth Factor Induces Tyrosine Phosphorylation, Membrane Insertion, and Activation of Transient Receptor Potential Channel 4

Dirk F Van Helden,Adam F Odell,Judith L Scott

doi:10.1074/jbc.m503646200

Dirk F Van Helden, Adam F Odell + Show 1 more

Open Access

https://doi.org/10.1074/jbc.m503646200

Copy DOI

Abstract

Various members of the canonical family of transient receptor potential channels (TRPCs) exhibit increased cation influx following receptor stimulation or Ca(2+) store depletion. Tyrosine phosphorylation of TRP family members also results in increased channel activity; however, the link between the two events is unclear. We report that two tyrosine residues in the C terminus of human TRPC4 (hTRPC4), Tyr-959 and Tyr-972, are phosphorylated following epidermal growth factor (EGF) receptor stimulation of COS-7 cells. This phosphorylation was mediated by Src family tyrosine kinases (STKs), with Fyn appearing to be the dominant kinase. In addition, EGF receptor stimulation induced the exocytotic insertion of hTRPC4 into the plasma membrane dependent on the activity of STKs and was accompanied by a phosphorylation-dependent increase in the association of hTRPC4 with Na(+)/H(+) exchanger regulatory factor. Furthermore, this translocation and association was defective upon mutation of Tyr-959 and Tyr-972 to phenylalanine. Significantly, inhibition of STKs was concomitant with a reduction in Ca(2+) influx in both native COS-7 cells and hTRPC4-expressing HEK293 cells, with cells expressing the Y959F/Y972F mutant exhibiting a reduced EGF response. These findings represent the first demonstration of a mechanism for phosphorylation to modulate TRPC channel function.

Highlights

Important components of this ubiquitous Ca2ϩ signaling pathway and represent an important area for potential drug therapy [7]
An immunoblot was performed on whole cell lysates from control, FLAG-human TRPC4 (hTRPC4), and YFP-tagged FLAG-hTRPC4 transfected COS-7 cells to demonstrate the ability of the anti-TRPC4 antibody to recognize transfected hTRPC4 and endogenous TRPC4
The findings presented here provide evidence that hTRPC4 undergoes rapid tyrosine phosphorylation mediated by Src family tyrosine kinases (STKs) following activation of the epidermal growth factor (EGF) receptor

Summary

Introduction

Important components of this ubiquitous Ca2ϩ signaling pathway and represent an important area for potential drug therapy [7]. Evidence for a role of TRPC channels in receptor-operated Ca2ϩ entry include the finding that isolated endothelial cells from the TRPC4 knock-out mouse exhibited reduced Ca2ϩ entry in response to acetylcholine, ATP, and thrombin application, and antisense oligonucleotide down-regulation of TRPC4 reduced Ca2ϩ oscillations in response to carbachol application in HEK293 cells (8 –10). Both store-operated and receptor-operated mechanisms can contribute to the activation of TRPC channels, in particular, TRPC4; the precise signaling components remain largely unknown. Dynamic regulation of the interaction between TRPC4 and NHERF may provide a mechanism for controlling TRPC4 surface expression and channel activation

Methods

Results

Conclusion